Adipocyte lipolysis contributes significantly to the pathogenesis of obesity-associated diseases by increasing levels of circulating free fatty acids (FFA). FFA promote insulin resistance and type 2 diabetes. My laboratory's long-term goal is to elucidate molecular mechanisms of lipolysis regulation. The proposed studies will investigate structure / function relationships of Perilipin A (Peri A), a lipid droplet- associated phosphoprotein that regulates lipolysis mediated by hormone sensitive lipase (HSL) and non-HSL lipase(s). Peri A acts dually as a suppressor of basal lipolysis (in the absence of hormonal stimulation) and as a potent enhancer of protein kinase A (PKA)-stimulated lipolysis (in the presence of hormonal stimulation). Despite its important regulatory role, the primary sequences and the mechanism(s) by which Peri A regulates lipase actions have not been determined. Our preliminary studies indicate that Perilipin regulates lipolysis via multiple regulatory domains, which exhibit surprising lipase specificity. The proposed studies will 1) identify the minimal domains of Peri A that modulate basal and PKA-stimulated lipolysis by HSL and non-HSL lipase(s), 2) determine the relative role of PKA phosphorylation sites in PKA- stimulated lipolysis by HSL and non-HSL lipase, and 3) define the in vivo effects of altered Peri A expression, Peri A truncations and Peri A PKA site mutants using Peri A transgenic and Peri null mice. Our adipocyte and systemic studies will measure basal lipolysis, lipolytic response to beta-adrenergic agents, and antilipolytic response to insulin. These studies will provide in vivo proof of concept tests of how Peri A expression levels, regulatory domains, and phosphorylation sites regulate basal and stimulated lipolysis. These data will be directed to the prevention and treatment of diabetes, hyperlipidemia and other obesity - associated disorders.